Sunitha Nagrath, Lecia V. Sequist, Shyamala Maheswaran, Daphne W. Bell, Daniel Irimia, Lindsey Ulkus, Matthew R. Smith, Eunice L. Kwak, Subba Digumarthy, Alona

Muzikansky, Paula Ryan, Ulysses J. Balis, Ronald G. Tompkins, Daniel A. Haber & Mehmet Toner

Nature December 2007

Presented by:

Katherine Choi and Linda Fong

Isolation of rare circulating tumor cells in cancer patients by microchip

technology

90% of Cancer Patient Deaths Result from Metastasis

Circulating tumor cells have emerged as a potential “surrogate biopsy” for metastatic disease.

There is a growing need for noninvasive methods, such as capturing these cells, to diagnose and monitor cancer.

Previous studies have suggestedthat the presence of circulating tumor cells in patients with metastatic carcinoma is associated with short survival.

Focusing on CTCs: a Diagnostic Cancer Target Circulating Tumor Cells Viable tumor-derived

epithelial cells Metastatic precursor cells or

cancer stem cells Rare : one per billion

haematologic cells 

Further discovery of cancer stem cell biomarkers and expand understanding of metastasis.

Presence of CTC is a strong prognostic factor for overall survival.

The “CTC-chip” Efficient and selective separation of viable

CTCs from peripheral whole blood samples. No pre-labelling or processing of blood.

Target CTCs interact with anti-EpCAM-coated microposts. (epithelial cell adhesion molecule)

Designing Chip Structure

Provides the specificity for CTC capture from unfractionated blood.

Micropost array of equilateral triangle geometry made chemically functional with anti- Ep-CAM

Optimizing CTC capture: Experimental Setup Offset: increases

probability of collision of cells with microposts by forcing cells to change their trajectory

Conclusion: Choose flow rate of 1-2 ml/h

Shear force: must be sufficiently low to favor cell-micropost attachment

Flow velocity: maximize frequency of cell-micropost contact.

CTC-Chip Capture Efficiency is Independent of Ep-CAM Expression Levels

Compared capture rates across: Concentrations of 2,000 Ag/cell to greater than

500,000 Ag/cell Results: Mean capture yield > 65% in all cases

Chip captures CTCs with equal efficiency across a wide range of Ep-CAM

Prostate breast bladder NSCLC

CTCs Isolated Directly From Whole Blood Without Need For Pre-Processing

Compare capture rates across: 50 to greater than 50,000 tumor cells per ml of

whole blood Results: Recovery rates > 60% in all cases

Lysed blood exhibits similar capture rates to whole blood

Chip captures with equal efficiency across varying concentrations

No blood sample pre-processing required.

CTC-chip Performance Across Clinical Cancer Samples

Epithelial cancers: NSCLC Prostate

Metastatic Localized

Breast Pancreatic Colon

Significant Gains in Purity and Sensitivity

CTCs only captured in patient samples with cancer

Sensitivity: CTCs detected in 99.1% of cancer samples

Purity: CTCs constitute ~50% of captured cells

CTC correlation with clinical response to treatment

Direct correlation between percent change in CTC quantity and percent change in tumor size

CTC-chip vs. Existing technology

Cancer Immunomagnetic Bead Purification

CTC Chip

Prostate 4±24 (average # of CTCs ± s.d.)

86±78 (metastatic), 94±63 (localized)

Lung 11±118 155±236

Breast 10±33 79±52

Colorectal

1±2 121±127

Pancreatic

1±2 196±228

Purity 0.01-0.1% 49%-67%

Yield ~20-60% 99.1%CTC-Chip outperforms the current leading technology for

identifying CTCs

Subsequent Molecular Analysis Cells are viable after capture Potential for immunostaining Tested the expression of 2 tumor specific markers PSA

and TTF-1

Advantages of CTC-Chip Technology Blood does not need preprocessing Unprecedented purity of CTC capture Sensitivity of chip detects CTC in cancer

patients. Detects CTCs across a wide range of cancers Applicable for capture of other rare circulating

cells via alternate Abs on microposts

Cancer

Immunomagnetic Bead Purification

CTC Chip

Prostate

4±24 (average # of CTCs ± s.d.)

86±78 (metastatic), 94±63 (localized)

Future Directions: CTCs found in localized prostate cancer and

metastatic prostate cancer are similar—novel finding with this technique and warrants further study

Questions?

Patient Population

68 Patients with epithelial cancers. 7 of 26 subjects with prostate cancer had untreated

clinically localized disease. Specimens collected before prostatectomy with curative intent.

Volume of blood: 2.7 mls/sample (range, 0.9 - 5.1 mls ) 20 healthy individuals as controls (3.0 ± 0.4 mls).

Type of Cancer Number of Samples

NSCLC 55

Prostate 26

Pancreatic 15

Breast 10

Colon 10

Total 116

Cancer Drug TreatmentCancer Drug Treatment

d. NSCLC 1st-line carboplatin, paclitaxel

e. NSCLC 2nd-line pemetrexed

f. Colorectal 1st -line 5FU, irinotecan

g. Pancreatic 1st-line gemcitabine, bevacizumab

h. Pancreatic 1st-line gemcitabine

i. Pancreatic 1st-line gemcitabine, erlotinib

Cancer Drug Treatment

a. Colorectal Cancer

1st-line infusional 5FU, oxaliplatin, and bevacizumab.

b. NSCLC 1st-line carboplatin, paclitaxel, and an experimental agent.

c. Esophageal cancer

1st-line cisplatin and irinotecan.

Purity of Capture – CTC chip

Average purity of capture (ratio of cytokeratin+ to CD45+ cells)

NSCLC 52%

Metastatic prostate 49%

Localized prostate 53%

Pancreatic 53%

Breast 60%

Colon 67%

Experimental Setup

Micropost Structure Array of 78,000 microposts within a 970 mm2

surface. Equilateral triangle configuration

PBS + varied EpCAM expression NSCLC NCI-H1650

and breast cancer SKBr-3 cells >500,000

Prostate cancer PC3-9 cells ~50,000

Bladder cancer T-24 cells ~2,000

Spiked into PBS at concentration of 100 cells/ml

> 65% mean capture yield

T-24 cells were captured as efficiently as high-level antigen-expressing cells, they believe due to augmented cell-substrate interations inherent within the CTC-chip.

Varied Blood Concentrations NCI-H1650 cells

spiked into whole blood from healthy donors

Concentrations from 50-50,000 tumor cells/ml

>60% recovery rate

White = CTCs, green = leukocytes.

a. Non functionalized control deviceb. Cell capture func with antiEpCAMc. Higher magnification of b.